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The Scientific World Journal
Volume 2012, Article ID 609597, 9 pages
http://dx.doi.org/10.1100/2012/609597
Research Article

Localisation of Abundant and Organ-Specific Genes Expressed in Rosa hybrida Leaves and Flower Buds by Direct In Situ RT-PCR

1Faculty of Natural Sciences, Institute for Ornamental and Woody Plant Science, University of Hannover, Herrenhauser Street 2, 30419 Hannover, Germany
2Department of Ornamental Plants, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences, Nowoursynowska 166, 02-787 Warsaw, Poland

Received 28 October 2011; Accepted 20 December 2011

Academic Editors: Y. Shoyama, M. Simmonds, and B. Vyskot

Copyright © 2012 Agata Jedrzejuk et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by using in situ RT-PCR in Rosa hybrida flower buds and leaves. As target genes, we used the abundant β-actin and RhFUL gene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.